1. Field of the Invention
The present invention relates to the field of detection assays using fluorescent detection reagents. More particularly, the invention relates to fluorescent immunoassays, such as those carried out by flow cytometry.
2. Description of Related Art
Particle analyzers, such as flow and scanning cytometers, are well known in the art. In these systems, fluorescently labeled particles, such as molecules, analyte-bound beads, or individual cells, are individually analyzed by exposing each particle to an excitation light, typically one or more lasers, and measuring the resulting fluorescence from each of dye labels. Each particle may be labeled with a multiplicity of spectrally distinct fluorescent dyes. Typically, detection is carried out using a multiplicity of photodetectors, one for each distinct dye to be detected. Both flow and scanning cytometers are commercially available from, for example, BD Biosciences (San Jose, Calif.).
In flow cytometers and other instruments that employ a multiplicity of photodetectors to detect a multiplicity of dyes, the collected light is separated into specific ranges of wavelengths, typically by a system of frequency-dependent filters and dichroic mirrors, such that the light detected by a particular photodetector is limited to a predefined range of wavelengths, referred to as a detection channel. The detection channels and dyes are selected such that the peak of the emission spectrum of each dye is within the frequency range of a different detection channel, i.e., each detection channel detects primarily the emission from a single dye. However, because of the breadth of the emission spectra of fluorescent dyes, typically a dye will fluoresce in more than one detection channels and, thus, measurements of dye fluorescence are not independent. The emission of one dye in detection channels intended for the detection of other dyes is referred to by a number of terms, such as spillover, spectral overlap, and crosstalk.
Methods of decreasing the effect of spectral overlap on dye fluorescence measurements are known in the art. Such methods involve adjustment of the signal measured by each photodetector by an amount calculated to compensate for the contribution from dyes other than the primary dye to be detected. Examples in the field of flow cytometry include Bagwell et al., 1993, “Fluorescence Spectral Overlap Compensation for any Number of Flow Cytometer Parameters”, Ann. N.Y. Acad. Sci. 677: 167-184; Roederer et al., 1997, “Eight Color, 10-Parameter Flow Cytometry to Elucidate Complex Leukocyte Hetrogeneity”, Cytometry 29: 328-339; and Bigos et al., 1999, Cytometry 36: 36-45; Verwer, 2002, BD FACSDiVa™ Option for the BD FACSVantage SE Flow Cytometer White Paper, and U.S. Pat. No. 6,897,954; each incorporated herein by reference. WinList™ (Verity Software House, Topsham, Me.) and FlowJo 5.7.2 software (Tree Star, Inc., Ashland, Oreg.) are a stand-alone software packages that allow software compensation on stored data files produced by a flow cytometer.
Typically, the amount of fluorescence spectral overlap compensation required is determined experimentally using compensation control beads, single-color particles dye with one of the fluorescent dyes used in the assay. The fluorescence signal of each bead is measured in each of the channels, which directly provides a measure of the spectral overlap into each of the channels. Compensation control beads are intended to match the spectral characteristics of the detection reagent labeled with the same dye. However, the spectral properties of pre-dyed particles may provide only an approximation of the spectral properties of the detection reagent when used in a assay.
A preferred method of measuring spectral overlap of fluorescently labeled antibody reagents into each of the detection channels is using BD™ CompBeads compensation particles (BD Biosciences, San Jose, Calif.). The particles, which are coated with anti-Ig antibodies, are combined with a fluorescently labeled antibody reagent, which becomes captured on the surface of the bead, to produce a particle labeled with the fluorescent dye. The spectral overlap of the dye is determined by measuring the emission of the labeled particle in each of the detection channels. The measurement typically is made relative to the emission from the unlabeled particle. The use of the assay detection reagent itself to stain the single-color compensation control particle minimizes differences in spectral overlap between the compensation control particles and the assay reagent as used in the assay.
BD CompBeads compensation particles are not used directly with prepackaged multicolor reagents that comprise multiple fluorescently labeled detection reagents in a single container because each compensation particle needs to be stained with only one of the detection reagents, and, thus, the detection reagents cannot be mixed prior to staining the compensation particles. Although additional vials of each single-color detection reagent that comprise multicolor reagent could be provided to allow the use of BD CompBeads compensation particles, this would largely negate the advantages of providing premixed multicolor reagents.